Affiliations: Department of Biomedical Science, College of Life Science, CHA University, Seoul 13488, Republic of Korea, Department of System Cancer Science, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, Seoul 10408, Republic of Korea

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Abstract

Lymphatic endothelial cells (LEC) are major components of the tumor microenvironment and, due to the relative leakiness of lymphatic vessels compared with blood vessels, are essential for tumor dissemination and metastasis. In the present study, small interfering RNA‑mediated suppression of E26 transformation‑specific domain‑containing protein Elk‑3 (ELK3) inhibited the proliferation, migration and tube‑forming ability of LEC. Suppression of ELK3 decreased vascular endothelial‑cadherin expression levels and increased the phosphorylation of β‑catenin. Furthermore, vascular endothelial growth factor receptor‑3 (VEGFR‑3) mRNA and protein expression levels were suppressed by the transfection of LEC with siELK3. As VEGFR‑3 serves a major role in lymphangiogenesis, ELK3 may be a novel therapeutic target to inhibit tumor dissemination through the lymphatic system.

Introduction

E26 transformation-specific (ETS) domain-containing
protein Elk-3 (ELK3) is a ternary complex factor belonging to the
ETS transcription factor family. ELK3 was first identified as a
transcriptional repressor of c-Fos, but it is able to function as a
transcriptional activator once phosphorylated by the
Ras/mitogen-activated protein kinase signaling pathway (1–3). The
transcriptional activity of ELK3 has been linked to vasculogenesis
and wound healing in cell lines and in mice (4–6). ELK3
regulates vascular integrity through early growth response protein
1 (4), and regulates angiogenesis
through the control of vascular endothelial growth factor (VEGF)
expression (6). Mice lacking ELK3
protein develop smaller tumors, which are not able to become
vascularized and oxygenated, indicating that ELK3 serves a major
role in tumorigenecity (6). In
addition to phosphorylation, the transcriptional activity of ELK3
is regulated by nuclear-cytoplasmic shuttling in response to
specific signaling pathways (7). ELK3
has two nuclear localization signals and it is primarily localized
to the nucleus under physiological conditions (7). ELK3 also has a nuclear export signal,
and the nuclear exclusion of ELK3 occurs via specific signaling
pathways, including the c-Jun kinase pathway (7).

The tumor and organ microenvironment is important
for cancer growth and metastasis. Diverse cells residing in the
microenvironment influence cancer cells through secreted factors
and their corresponding signaling pathways (8). These cell-to-cell communications
contribute to cancer progression, angiogenesis, invasion,
epithelial-to-mesenchymal transition phenotypes and antitumor drug
resistance (9–11). Therefore, cells within the tumor
microenvironment have emerged as attractive targets to effectively
inhibit cancer. Lymphatic vessels (LV) are major components of the
tumor microenvironment and are composed of lymphatic endothelial
cells (LEC) (12). As LVs are more
permeable than blood vessels, they are particularly important for
tumor dissemination and metastasis (12). Despite the importance of LV in tumor
progression, the factors secreted by LEC and the signals through
which they influence the behavior of the tumor remain to be
elucidated.

The present study demonstrated that ELK3 regulates
cell migration, tube formation and cell permeability of LEC. These
results indicate that ELK3 is a major regulator of
lymphangiogenesis in LEC.

The non-specific control siRNA (siNS; #D-001810-10;
Dharmacon, Inc., Chicago, IL, USA) and human ELK3 siRNA (siELK3;
#L-010320-00-0005; Dharmacon, Inc.) were obtained from Dharmacon
(GE Healthcare Life Sciences, Chalfont, UK). The siRNAs (100 nM)
were transfected into LEC using Lipofectamine® 2000
(Thermo Fisher Scientific, Inc.) according to the manufacturer's
protocol, and cells were collected at 48 h following transfection
for further analysis. Total cellular RNA was extracted using
TRIzol® reagent (Thermo Fisher Scientific, Inc.,
Invitrogen, Carlsbad, CA, USA) following the manufacturer's
protocol. Total RNA (2 µg) was used for single-stranded cDNA
synthesis with OmniScript® reverse transcriptase (Qiagen
GmbH, Hilden, Germany). RT-qPCR was performed using the CFX96
Touch™ Real-Time PCR Detection system (Bio-Rad Laboratories Inc.,
Hercules, CA, USA) using SYBR-Green I (Qiagen Inc., Valencia, CA,
USA). The final volume of PCR mixture was 20 µl, comprising 1 µg
cDNA, 10 pmol forward and reverse primers and 10 µl of 2X SYBR mix.
The reactions were performed for 40 cycles using optimized cycling
conditions (denaturation at 95°C for 1 min, annealing at 55°C for
30 sec and extension at 72°C for 30 sec). RT-qPCR data was
processed based on the 2−ΔΔCq method (13). The primers used in this analysis are
listed in Table I.

Protein extraction and western
blotting

Total protein was isolated using Cell Lysis buffer
(#9803; Cell Signaling Technology Inc., Danvers, MA, USA) according
to the manufacturer's protocol. A bovine serum albumin (BSA)
standard curve was used to estimate the protein concentration. A
total of 8 BSA standards were made by dilating 2 µg/µl albumin
standard (#23209; Thermo Fisher Scientific, Inc.) into cell lysis
buffer, and they were then incubated at 37°C for 30 min. A standard
curve was produced based on the absorbance values of BSA solution
in plate reader (Epoch microplate spectrophotometer; Biotek
Instruments, Inc., Winooski, VT, USA) at 562 nm. Total protein (20
µg) was separated by 10% SDS-PAGE and transferred to a
polyvinylidene difluoride (PVDF) membrane (EMD Millipore,
Billerica, MA, USA). The membrane was blocked by 5% skim milk
solution at room temperature for 30 min and then incubated with an
anti-ELK3 antibody (#sc-17860, 1:1,000 dilution; Santa Cruz
Biotechnology Inc., Dallas, TX, USA), an antibody recognizing
active β-catenin (unphosphorylated at Ser33/37/Thr41, #4270S), an
anti-phospho-β-catenin antibody (phosphorylated at Ser33/37/Thr41,
#9561S) (both from Cell Signaling Technology, Inc.), an anti-lamin
B antibody (#sc-6216) or an anti-β-actin antibody (#sc-47778) (both
from Santa Cruz Biotechnology, Inc.) overnight at 4°C, followed by
incubation with a secondary antibody (#sc-2005, 1:2,000 dilution;
Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The
membrane was washed three times in TBST for 5 min each time.
Immunoreactive proteins were detected using the SuperSignal West
Pico Chemiluminescent Substrate Western Blot Detection system
(catalog no. 34080; Thermo Fisher Scientific, Inc.) according to
the protocol of the manufacturer. All experiments were repeated at
least three times.

2D migration assay

The migration potential of LEC was assessed using a
wound healing assay. LEC, following 24 h transfection with siNS or
siELK3, were cultured on 26×76 mm glass coverslips in endothelial
cell growth medium-2 supplemented with 5% FCS and wounded using a
micropipette tip when the cells were fully confluent. The cells
were incubated for 24 h at 37°C and then observed for migration
using a light microscope (CKX41SF; Olympus, Tokyo, Japan).

In vitro tube formation assay

The ability of LEC to form capillary-like structures
(tubes) was assessed using a tube formation assay (14). LEC were harvested and cultured in
media with or without recombinant VEGF-C for 30 min at room
temperature. A total of 1.5×104 LEC cells were seeded on
slides coated with Matrigel (#356231; Corning Inc., Corning, NY,
USA) supplemented with vitronectin (SPR3186; Sigma-Aldrich; Merck
KGaA) (5 mg/ml). Cells were incubated for 24 h in the presence of
5% CO2 at 37°C for further analysis.

Cell proliferation assay

Cell proliferation was analyzed using the MTT assay.
Briefly, 3×103 cells were seeded onto 96-well plates and
cultured for 24, 48 and 72 h at 37°C. MTT solution was added to
each well at a final concentration of 0.5 mg/ml and incubated for 4
h at 37°C. The resulting formazan crystals were dissolved in 150 µl
dimethyl sulfoxide per well. The absorbance was measured at a
wavelength of 570 nm using an automated plate reader (Thermo Fisher
Scientific, Inc.).

Analysis of VEGF-C protein in the
culture media

The amount of VEGF-C in the culture media was
measured by VEGF-C Quantikine ELISA kit (R&D Systems, Inc.,
Minneapolis, USA; #DVEC00) according to the protocol of the
manufacturer.

Statistical analysis

Graphical data are presented as the means ± standard
deviation. P<0.05 and P<0.01 were considered to indicate
significant and highly significant results based on Student's
t-test analyses, respectively. Statistical analyses were performed
using the SAS statistical package v.9.13 (SAS Institute, Cary, NC,
USA; http://www.sas.com/).

Results

ELK3 regulates the proliferation of
LEC

ELK3 is highly expressed in the LV of mice (13). The expression of ELK3 in LEC was
examined to elucidate the role of ELK3 in lymphangiogenesis. The
expression levels of ELK3 in LEC were comparable to those in HUVEC
cells and in the MDA-MB-231 and MCF-7 breast cancer cell line, in
which ELK3 has been established to be important in angiogenesis
(Fig. 1A). ELK3 is primarily
localized to the nucleus unless it is actively exported to the
cytoplasm via specific signaling molecules, including c-Jun
(16). As presented in Fig. 1B, the phosphorylated form of ELK3 was
detected in the nucleus, indicating that ELK3 may function as an
active transcription factor in LEC. To examine the role of ELK3 in
LEC, the protein expression of ELK3 was suppressed using siRNA
(Fig. 1C). Suppression of ELK3
protein expression did not exhibit any effect on LEC morphology but
did result in a decreased proliferation rate (P<0.05; Fig. 1D).

ELK3 regulates migration and tube
formation of LEC

An in vitro scratch assay that mimics cell
migration into an artificial wound produced on a cell monolayer was
used to investigate the role of ELK3 in LEC migration (17). The healing ability of LEC was
inhibited at 24 h following wounding in cells transfected with
siELK3 (Fig. 2A). The effect of ELK3
suppression on the vascular behavior of LEC was evaluated using a
tube formation assay. As presented in Fig. 2B, LEC transfected with siELK3 formed
fewer branch points compared with those in the siNS controls in the
presence and absence of VEGF-C (*P<0.05, **P<0.01). However,
tube length was not affected by the silencing of ELK3 (Fig. 2C). These results suggest that ELK3 may
regulate the vasculogenic activity of LEC.

Expression of VE-cadherin and VEGFR-3
and the phosphorylation of β-catenin, are regulated by ELK3

Endothelial permeability is closely associated with
the dissemination of cancer cells and is, therefore, particularly
important in cancer biology (18). As
the expression of VE-cadherin regulates endothelial permeability
and mediates cell-to-cell contact (19), the expression levels of VE-cadherin
were analyzed in the presence and absence of siELK3. As presented
in Fig. 3A, the expression of
VE-cadherin mRNA was decreased following transfection with siELK3
(P<0.05). The accumulation of VE-cadherin near the membrane and
at points of cell-to-cell contact was lower in siELK3-transfected
LEC compared with siNS control cells (P<0.05, Fig. 3B). These results suggest that ELK3 may
function as a positive regulator of VE-cadherin expression levels
in LEC. In addition to the expression of VE-cadherin, the
phosphorylation of β-catenin has also been implicated in
VE-cadherin-mediated cell adhesion (20–22). As
the phosphorylation of β-catenin correlates with the loss of
VE-cadherin function (23), the
phosphorylation of β-catenin was compared in siELK3-transfected and
control LEC in the presence and absence of VEGF-C. Notably, ELK3
suppression was correlated with the phosphorylation of β-catenin in
the presence and absence of VEGF-C (Fig.
3C). These results suggest that ELK3 may regulate the
transcriptional expression of VE-cadherin as well as the
phosphorylation of β-catenin.

In the tumor microenvironment, particularly in
breast cancer, LV is a predominant route of tumor dissemination
(24). Tumor lymphangiogenesis is
driven by growth factors, including VEGF-C and VEGF-D, which are
secreted from tumor cells (25). LEC
in the LV also secrete factors into the tumor microenvironment that
facilitate tumor dissemination (24).
Therefore, the effect of ELK3 suppression on the expression levels
of angiogenic factors was analyzed by RT-qPCR. VEGFR-3, which
serves an important role in lymphangiogenesis as the receptor for
VEGF-C, was significantly downregulated, whereas the expression of
VECF-C was upregulated in siELK3-transfected LEC (Fig. 3D). Consistent with the mRNA levels,
the quantity of VEGF-C protein in the culture media of
siELK3-transfected LEC was higher compared with the controls
(P<0.05, Fig. 3E). These results
demonstrate that ELK3 suppression may make LEC insensitive to
VEGF-C stimulation, possibly via the reduced expression of
VEGFR-3.

Discussion

The present study demonstrated that ELK3 may be
involved in the proliferation, migration and tube formation by LEC,
activities that are required for lymphangiogenesis (14). In particular, the results suggested
that ELK3 positively regulates VEGFR-3 expression in LEC. Other ETS
family transcription factors possess the ability to induce
lymphangiogenesis by regulating VEGFR-3 (26). However, the current study, to the best
of our knowledge, is the first report that ELK3 may regulate
VEGFR-3 expression levels.

The lymphatic endothelium functions as a selective
permeable barrier controlling transfer between vessel and tissues
(27). Impaired endothelium
permeability results in persistent vascular leakage, and is
implicated in diverse pathological conditions (28). Mice that express a mutant version of
ELK3 lacking the ETS DNA-binding domain develop dilated LV
(4). This is concordant with the
results of the current study, which suggest that ELK3 may be
implicated in LEC permeability. It is also important to evaluate
whether VEGFR-3 downstream signaling is regulated by ELK3, as the
binding of ligands including VEGF-C to VEGFR-3 activates the
phosphoinositide 3-kinase/protein kinase B (PI3-K/Akt) signaling
pathway, and the physical interaction between VEGFR-3 and PI3 K is
associated with lymph node metastasis (29).

In conclusion, it is therefore important for future
studies to elucidate whether alterations in ELK3 expression are
associated with disturbances in PI3-K/Akt signaling. As the
VEGFR-3/PI3-K signaling pathway is implicated in lymphangiogenesis
in the LEC, the ELK3-VEFGR3-PI3K axis may be a novel therapeutic
target to inhibit tumor dissemination through the lymphatic
system.

Acknowledgements

The present study was supported by the Korea Science
and Engineering Foundation of the Korean government (grant no.
2015R1A2A2A01003498). Ministry of Education, Science, and
Technology (NRF-2017-M3A9B4031169).